One function of ubiquitin, a highly conserved 76-residue protein, is to serve as a signal for degradation of conjugated acceptor proteins. More recent work [1] has identified another,"chaperone" function of ubiquitin, in which its covalent association with other proteins promotes formation of specific cellular structures [1]. Studies of the last several years, and in particular studies supported by the present grant, have demonstrated the involvement of ubiquitin-dependent processes in a remarkably broad range of cellular functions, from protein degradation and maturation to cell cycle control, stress response and DNA repair. The objective of the research described in this renewal application is to advance the understanding of the ubiquitin system. Specific Aims: 1) Molecular genetic analysis of the yeast (S. cerevisiae) Ub-activating enzyme (E1, encoded by the UBA1 gene), and the use of conditional ubal mutants to test Ub involvement in the degradation of specific short-lived proteins. 2) Molecular genetic analysis of the yeast Ub-conjugating enzymies (E2's, encoded by the UBC1-UBC8 genes), and identification of E2's that function in the N-end rule pathway of protein degradation. 3) Studies on Ub-related aspects of the yeast N-end-recognizing protein (E3) encoded by the UBR1 gene. 4) Molecular genetic analysis of the yeast Ub-specific proteases (encoded by UBP genes) that cleave Ub off its linear or branched conjugates with itself or other proteins. 5) Using non-deubiquitinatable Ub fusions (Ub-Pro-Betagal or (Ub-Val76)-Met-Betagal) to dissect a proteolytic pathway that recognizes a single Ub moiety as degradation signal and is distinct from the N-end rule pathway. 6) Construction of Ub bearing a peptide tag, and its use to detect and identify specific constitutive and stress-inducible Ub conjugates in S. cerevisiae. 7) Further biochemical and genetic analyses of the in vivo ubiquitination and degradation of the yeast MATAlpha2 repressor. 8) Cloning and functional analysis of mammalian (mouse) genes that encode Ub-related components of the N-end rule pathway.